Among the most common and critical health problems in the United States today is atherosclerosis, and its attendant complications, in particular, coronary heart disease. A number of risk factors have been implicated in the development of "premature" atherosclerosis, one of the most important of these being elevated plasma cholesterol. Because of the crucial role that cholesterol appears to play in the occurrence of heart disease, a great deal of attention has been devoted to the study of its metabolism in the human body.
Of particular recent interest is the investigation of the relationship between the levels of plasma lipoproteins or serum lipids and the risk of development of coronary heart disease. Both high density lipoproteins (HDL) and low density lipoproteins (LDL) are carriers of cholesterol in the form of cholestryl esters. There is some indication, however, that while LDL cholesterol is a positive risk factor (Kannel et al., Ann Intern Med 90:85-91, 1979), HDL is an even more important negative risk factor (see FIG. 1). Although the exact functions of these lipoproteins are not completely determined, it appears that HDL serves particularly to remove cholesterol from peripheral cells, and transport it back to the liver, where a large proportion of the cholesterol excreted from the body is removed.
One current idea on the specific roles of LDL and HDL in the development of cardiovascular disease emphasizes the role of the overloading of the lysosomes of the cells of the arterial walls with metabolites which are generally hydrolyzed rather slowly, specifically cholesteryl esters and triglycerides. These are transported from the liver and intestine by plasma LDL. Should the amount of these lipids exceed the capacity of the HDL for transporting them to the liver for excretion, cells in certain critical areas, such as the arterial wall, become gorged with cholestryl esters. This overloading eventually results in impaired cell function, and, if continued, cell death. The continued overloading further results in the accumulation of cellular debris, and the formation of atherosclerotic plaque in the vessel wall. This in turn may lead to blockage of the artery, and spasms of the muscular layer, events which may manifest themselves as coronary heart disease or strokes.
Each of the known plasma lipoproteins is formed by the association of one or more apoprotein moieties with phospholipid. Considerable attention has been given in recent years both to the role of the apoproteins in the overall function of the lipoprotein, and to its manner of association with the lipid. There is much evidence to suggest that the protective effect of HDL may be due to participation in the process of reverse cholesterol transport, which is in turn dependent upon the levels of the major HDL apoprotein component, A-I. The latter has the effect of stimulating lecithin:cholesterol acyl transferase activity, which is important in concentrating cholestrol, in the form of cholesteryl ester, inside the HDL particles.
A mechanism to explain certain features of protein lipid interactions in the plasma lipoproteins has been suggested in the amphipathic helix hypothesis (Segrest et al., FEBS Letter, 38:247-253, 1974). This model suggests a general structural arrangement of amino acid residues which result in helical domains, called amphipathic helices, containing polar and non-polar faces. A general distribution of the charged residues was proposed, with the positive occurring along the interface between the polar and non-polar faces, and the negative along the center of the polar face. This arrangement of the charged residues allows the lysine or arginine acyl side chains to contribute to the hydrophobicity of the non-polar face. The charged residues also seem to form topographically close complementary ion pairs, the number of which may be significant. Further, this model allows for ionic interactions between positively charged side chains and the phosphate group of the phospholipid, as well as between negatively charged residues and positively charged groups on the phospholipid. Such interactions may play a role in initiating or contributing to the stability of the peptide-lipid complex.
Given this proposed model, it should theoretically be possible to attempt construction of synthetic analogs of apo A-I which are capable of functioning in much the same way as the model apolipoprotein. In order to act as a satisfactory substitute for APO A-I, a synthetic peptide would be required to (1) form small, stable, discoidal complexes with phospholipid, as apo A-I does normally in nascent HDL, and (2) stimulate lecithin-cholestryl acyl transferase (LCAT) activity. It should also preferably be able to displace native apolipoprotein, particularly APO A-I, from HDL complexes.
Recently, various synthetic functional apolipoprotein analogues have been reported. Sparrow, et al. (Peptides, Eds, Rich & Gross, p. 253-256, 1981) have produced a series of "Lipid Associating Peptides" (LAP) which have been shown to activate LCAT. Although it is assumed from this evidence that these peptides would have a high affinity for lipids, there are no numerical data which conclusively support this assumption. Kaiser and Kezdy (PNAS, USA 80:1137-1143; 1983; Science 223:249-251, 1984) also disclose amphipathic peptides which have been shown, to some extent, to mimic the activity of apo A-I. Kanellis et al. (Jour Biol. Chem. 255:11464-11472, 1980), and Segrest et al. (Jour Biol. Chem. 258:2290-2295, 1983) have described an amphipathic peptide, 18 As, which exhibit LCAT activation and the ability to displace native apolipoprotein from HDL complexes. None of these peptides, however, has been as yet shown to form stable, discoidal, nascent, HDL-like complexes with phospholipids, a feature which is critical to their utility as components of pharmaceutically useful synthetic lipoproteins.
It has now been discovered that a new series of peptides exhibit an unexpected improvement over previously known peptides in their ability to mimic apo A-I activity. These new peptides not only are capable of stimulating LCAT activity and displacing apolipoprotein from native HDL, but also displace a higher percentage of apolipoprotein than known amphipathic peptides, and are the first to demonstrate the capacity for forming compact, discoidal nascent HDL-like complexes. As noted above, the latter characteristic is particularly important to the peptides' contemplated role in the treatment of atherosclerosis by the administration of synthetic HDL complexes.